Viologen linked acridine based molecule and process for the preparation thereof

ABSTRACT

The present invention provides a series of novel bifunctional molecules based on viologen linked acridine, bisacridine and bisacridinium salts of the general formula 1 (1a, 1b, 1c and 1d) and or pharmaceutically acceptable derivatives thereof, which can be used as phototherapeutic and catalytic photoactivated DNA cleaving agents. These molecules are very stable and exhibit high solubility in buffer at physiological conditions. They undergo strong binding with DNA through intercalation and groove binding interactions and show remarkably high affinity for poly(dA).poly(dT) sequences. Upon photoactivation, they cleave DNA catalytically and selectively at guanine (G) sites in duplex DNA exclusively through cosensitization mechanism with a preference for 5′-G over 3′-G. They induce unusally high specificity of cleavage at G site of the AG two base bulge sequences. Accordingly, the viologen linked acridine based molecules described herein are extremely useful as probes for DNA structures and catalytic photoactivated DNA cleaving agents in biological applications.

FIELD OF THE INVENTION

[0001] The present invention relates to viologen linked acridine basedmolecule of the general formula 1 (1a, 1b, 1c and 1d)

[0002] 1a wherein

Y=—(CH₂)_(m)— wherein n=1-11

R=—MV²⁺—(CH₂)_(m)—CH₃2X^(Θ) or

—Pyr²⁺—(CH₂)_(m)—CH₃2X^(Θ) wherein m=1-13

[0003] 1b wherein

Y=—(CH₂)_(n)— wherein n=1-11

R=—MV²⁺—(CH₂)_(m)—Acr 2X^(Θ) or

—Pvr²⁺—(CH₂)_(m)—Acr 2X^(Θ) wherein m=1-11

[0004] 1c wherein

Y=ortho or para tolyl

R=—MV²+—(CH₂)_(m)—CH₃2X^(Θ) or

—Pyr²⁺(CH₂)_(m)—CH₃2X^(Θ) wherein m=1-13

[0005] 1d wherein

Y=—(CH₂)_(n)— wherein n=1-10

R=—Acr⁺—R¹X^(Θ)/2X^(Θ)

[0006] wherein N in the acridine main ring is also quarternised by alkylgroup

R¹=—(CH₂)_(m)—CH₃ and —(CH₂)_(m)—C₆H₄—(CH₂)_(m)—(para),

[0007] wherein

m=0-13

[0008]

Formula 1

[0009] and/or pharmaceutically acceptable derivatives thereof, useful asphototherapeutical and catalytic photoactivated DNA cleaving agents. Thepresent invention also relates to a process for the preparation of thenovel molecule of formula 1. The novel molecules of the invention areuseful for the stabilization of DNA including duplex, triplex andquadruplex structures through intercalation and/or bisintercalation andgroove binding interactions. The novel molecules of the invention arealso for the catalytic photoactivated cleavage of DNA purely throughcosensitization with selectivity at guanine (G) sites in duplex and AGtwo base bulge containing sequences.

[0010] The present invention also relates to a series of bifunctionalmolecules of the general formula 1 (1a, 1b, and 1c) and derivativesthereof, are also useful as photocatalysts for the oxidation of water togenerate hydrogen in industrial applications.

BACKGROUND OF THE INVENTION

[0011] Design of functional molecules that bind selectively to nucleicacids (DNA or RNA) and are capable of cleaving duplex or single strandednucleic acids is an active area of research that has importantbiochemical and biomedical applications. Some of these effective agentshave been extremely useful in the treatment of various diseases and alsoas probes for understanding DNA structures and DNA-protein interactions.While natural restriction enzymes have been very useful in many of theseapplications, their large size and/or limited range of sequencerecognition capabilities prevent their general use. Hence, syntheticfunctional molecules that cause site-selective or sequence specificmodifications of DNA and offer a clean and efficient way of cutting DNAat sites that are not recognized by conventional restriction enzymes arehighly required.

[0012] In this context, a large number of synthetic ligands have beendeveloped, which have it ability to recognize and bind to specificsequences or structural domains in DNA and exhibit nucleolytic activityunder physiological conditions (chemical nucleases) or uponphotoactivation (photonucleases). Some of these include,1,10-phenanthroline-copper, ferrous-EDTA, bleomycin, enediyeneantibiotics and anthraquinones. For examples, references may be made toU.S. Pat. No. 5,985,557; No. 6,090,543; No. 5,739,022; No. 5,556,949;No. 5,552,278; No. 5,504,075; No. 4,942,227; Nielsen, P. E. J. Mol.Recog.. 1990, 3, 1; Papavassiliou, A. G. Biochem. J. 1995, 305, 345;Sigman, D. S.; Graham, D. R.; D'Aurora, V.; Stern, A. M. J. Biol. Chem.1979, 254, 12269; Pope, L. E.; Sigman, D. S. Proc. Natl. Acad. Sci. USA.1984, 81, 3; Tullius, T. D.; Dombroski, B. A. Proc. Natl. Acad. Sci.USA. 1986, 83, 5469; Hertzberg, R. P.; Dervan, P. B. J. Am. Chem. Soc.1982, 104, 313; Hecht, S. M.; Bleomycin: Chemical, Biochemical andBiological aspects, Ed., Springer Verlag: New York, 1979; Sausville, E.A.; Peisach, J.; Horwitz, S. B. Biochemistty, 1978, 17, 2740. However,synthetic ligands that are versatile and mimic the conventionalrestriction enzymes are yet to be developed.

[0013] Of the several classes of DNA cleaving systems reported, thephotoactivated cleaving agents have been found to posses significantpractical advantages over the reagents that cleave DNA under thermalconditions. An interesting aspect of the photoactivated DNA cleavingagent is that it allows the reaction to be controlled spatially andtemporally by combining all of the components of the reaction mixturebefore the irradiation. Excitation of the reaction mixture with anappropriate light source initiates the reaction, which continues untilthe light is shut off The ability to control light, in both spatial andtemporal sense would be advantageous for applications ranging from thetime resolved probing of various biochemical processes such astranscription and translation to genomic analysis and therapeuticagents. For selected examples, reference may be made to U.S. Pat. No.5,994,410; No. 5,734,032; No. 5,650,399; No. 5,607,924; No. 6,087,493;No. 6,057,096; 5,767,288: No. 5,439,794; Armitage, B. Chem. Rev. 1998,98, 1171 and references sited therein; Kochevar, I. E.; Dunn, D. A.Bioorg. Photochem. 1990, 1, 273 and references sited therein; Paillous,N.; Vicendo, P. J. Photochem. Photobiol. B 1993, 20, 203; Nielsen, P.E.; Jeppesen, C.; Buchardt, O. FEBS lett. 1988, 235, 122; Chow, C. S.;Barton, J. K. Methods Enzymol. 1992, 212, 219; Chang, C. -H.; Meares, C.F. Biochemistry 1982, 21, 6332; Riordan, C. G.; Wei, P. J. Am. Chem.Soc. 1994, 116, 2189; Thorp, H. H. Angew. Chem., Int. Ed. Eng. 1991, 30,1517; Armitage, B.; Yu, C.; Devadoss, C.; Schuster, G. B. J. Am. Chem.Soc. 1994, 116, 9847; Adam, W.; Cadet, J.; Dall'Acqua, F.; Epe, B.;Ramaiah, D.; Saha-Moller, C. R. Angew. Chem. Int. Ed. Engl. 1995, 34,107; Uesawa, Y.; Kuwahara, J.; Sugiura, Y. Biochem. Biophys. Res.Commun. 1989, 164, 903; Ito, K.; Inoue, S.; Yamamoto, K.; Kawanishi, S.J. Biol. Chem. 1993, 268, 13221; Saito, I.; Takayama, M.; Matsuura, T.;Matsugo, S.; Kawanishi, S. J. Am. Chem. Soc. 1990, 112, 883; Sako, M.;Nagai, K.; Maki, Y. J. Chem. Soc. Chem. Commun. 1993, 750.

[0014] These photoactivated cleaving agents found to cleave DNA (i) bygeneration of diffusible (singlet oxygen) and non-diffusible (hydroxylradicals) reactive intermediates, (ii) hydrogen atom abstraction and(iii) electron transfer. Most of the systems reported so far, initiatephotocleavage by more than one mechanism. Though the damage induced byall these mechanisms lead to the initial modification of either sugar ornucleobase, which then results in phosphodiester cleavage, seriousefforts are in progress to develop reagents which cleave DNA purely byone mechanism and also to target these cleaving agents to specificsequences or domains in DNA. References may be made to Cadet, J.;Teoule, R. Photochem. Photobiol. 1978, 28, 661; Croke, D. T.;Perrouault, L.; Sari, M. A.; Battioni, J. P.; Mansuy, D.; Magda, D.;Wright, M. M.; Miller, R. A.; Sessler, J. L.; Sansom, P. L. J. Am. Chem.Soc. 1995, 117, 3629; Theodorakis, E.; Wilcoxen, K. M. Chem. Commun.1996, 1927; Suenaga, H.; Nakashima, K.; Hamachi, I.; Shinkai, S.Tetrahedron Lett. 1997, 38, 2479; Cullis, P. M.; Malone, M. E.;Merson-Davies, L. A. J. Am. Chem. Soc. 1996, 118, 2775; Sies, H.;Schulz, W. A.; Steenken, S. J. Photochem. Photobiol. B 1996, 32, 97;Saito, I.; Takayama, M.; Sugiyama, H.; Nakamura, T. In DNA and RNACleavers and Chemotherapy of Cancer and ViraL Diseases; Meunier, B.,Ed.; Kluwer: Netherlands, 1996, pp 163-176.

[0015] Recently, there has been growing interest in designing molecules,which cleave DNA effectively through photoinduced electron transfermechanism involving purely by the oxidation of nucleobases. A uniquefeature of this mechanism is that one can have reasonable control overthe cleavage. It has been observed that DNA cleavage by this mechanismoccurs at guanine (G), since guanine is the most easily oxidizable baseof the nucleic acids because of its low ionization potential. A largenumber of organic as well as inorganic systems have been reported whichcause DNA cleavage by photoinduced electron transfer mechanism. However,most of these reagents were found to be less efficient with the cleavageefficiency in the order of 10⁻⁸. References may be made to Sevilla, M.D., D'Arcy, J. B.; Morehouse, K. M.; Englehardt, M. L. Photochem.Pholobiol. 1979, 29, 37; Blau, W.; Croke, D. T.; Kelly, J. M.; McConnel,D. J.; OhUigin, C.; Van der Putten, W. J. M. J. Chem. Soc. Chem. Commun.1987, 751; Sage, E.; Le Doan, T.; Boyer, V.; Helland, D. E.; Kittler, .;Hélène, C; Moustacchi, E. J. Mol. Biol. 1989, 209, 297; Brun, A. M.,Harriman, A. J. Am. Chem. Soc. 1991, 113, 8153; Ly, D.; Kan, Y.;Armitage, B.; Schuster, G. B. J. Am. Chem. Soc. 1996, 118, 8747: Hall,D. B.; Holmlin, R. E.;Barton, J. K. Nature 1996, 382, 731; Gasper, S.M.; Schuster, G. B. J. Am. Chem. Soc. 1997, 119, 12762. Therefore,efficient photoactivated DNA cleaving agents based on electron transfermechanism are highly desired for biological applications.

[0016] In the case of the photoactivated DNA cleaving agents by electrontransfer mechanism, the efficiency of the reaction depends on thereduction potential of the cleaving agent, excited state energy and theoxidation potential of the ground state base, in addition to otherconditions. For an efficient reaction to occur, the rate of the forwardelectron transfer from the donor to an acceptor must be greater than theback electron transfer process. Therefore, the inefficiency associatedwith the photoactivated DNA cleaving agents can be attributed to theexistence of an efficient back electron transfer between the resultantoxidized base and the reduced sensitizer. To overcome the drawback ofthe back electron transfer process associated with such systems, a fewexamples based on cosensitization mechanism have been developed (Dunn,D. A.; Lin, V. H.; Kochevar, I. E. Biochemistry 1992, 31, 11620;Atherton, S. J.; Beaumont, P. C. J. Phys. Chem. 1987, 91, 3993;Fromherz, P.; Rieger, B. J. Am. Chem. Soc. 1986, 108, 5361). Thesesystems consists of a sensitizer, which is also an intercalator,transfers an electron upon photoactivation to a cosensitizer (electronacceptor), bound on the surface of DNA. The photosensitization involvingthe cosensitizer that bound far away from the sensitizer is expected toinhibit the back electron transfer and thereby increase the DNAcleavage. However, in reality, only marginal improvement in DNA cleavageefficiency (in the order of 10⁻⁷) was observed in these systems owing tothe complications with respect to the concentration, distance and DNAbinding affinities of the sensitizer and cosensitizers.

[0017] Therefore, development of small compounds which are, soluble inaqueous medium, overcome the inefficiency due to the back electrontransfer, undergo strong binding interactions with DNA, selective andeffective in inducing DNA cleavage purely through electron transfermechanism are highly desired for biological applications including cleanand efficient way of cutting DNA at sites that are not recognized by theconventional restriction enzymes.

[0018] It is an objective of the present investigation to providefunctional molecules which bind strongly and selectively with DNA andact as selective and effective photoactivated DNA cleaving agents whichfunction purely through cosensitization mechanism.

OBJECTS OF THE INVENTION

[0019] The main objective of the present invention is to provide novelbifunctional molecules based on viologen linked acridines or derivativesthereof, which can be used as phototherapeutical and catalyticphotoactivated DNA cleaving agents.

[0020] Another objective of the present invention is to providebifunctional molecules based on viologen linked acridines or derivativesthereof, which can act as probes for various DNA structures (singlestrand, duplex, triplex and quadruplex) of biological significance andwith high selectivity.

[0021] Yet another objective of the present invention is to providebifunctional molecules based on viologen linked acridines or derivativesthereof, which can act as catalytic photoactivated DNA cleaving agentsof duplex and base bulges containing DNA, purely through cosensitizationmechanism.

[0022] Still another objective of the present invention is to providebifunctional molecules based on viologen linked acridines or derivativesthereof, which can act as photocatalysts for the oxidation of water inindustrial applications. SUMMARY OF THE INVENTION

[0023] Accordingly the present invention relates to viologen linkedacridine based molecule of the general formula 1 (1a, 1b, 1c and 1)below

[0024] 1a wherein

Y=—(CH₂)_(m)— wherein n=1-11

R=—MV²⁺—(CH₂)_(m)—CH₃2X^(Θ) or

—Pyr²⁺—(CH₂)_(m)—CH₃2X^(Θ) wherein m=1-13

[0025] 1b wherein

Y=—(CH₂)_(n)— wherein n=1-11

R=—MV²⁺—(CH₂)_(m)—Acr 2X^(Θ) or

—Pvr²⁺—(CH₂)_(m)—Acr 2X^(Θ) wherein m=1-11

[0026] 1c wherein

Y=ortho or para tolyl

R=—MV²+—(CH₂)_(m)—CH₃2X^(Θ) or

—Pyr²⁺(CH₂)_(m)—CH₃2X^(Θ) wherein m=1-13

[0027] 1d wherein

Y=—(CH₂)_(n)— wherein n=1-10

R=—Acr⁺—R¹X^(Θ)/2X^(Θ)

[0028] wherein N in the acridine main ring is also quarternised by alkylgroup

R¹=—(CH₂)_(m)—CH₃ and —(CH₂)_(m)—C₆H₄—(CH₂)_(m)—(para),

[0029] wherein

m=0-13

[0030]

[0031] The present invention also relates to a process for thepreparation of the novel bifunctional molecule based on viologen linkedacridines, bisacridines and acridinium salts of the general formula 1(1a, 1b, 1c and 1d) above, said process comprising forming a solution ofω-(acridin-9-yl)-α-bromoalkanes and/or 1-alkyl-4,4′-bipyridiniumbromides in dry acetonitrile in the ratio of 1:1, stirring the solutionat a temperature in the range of 20-50° C. for a time period in therange between 8-24 h to obtain a precipitate, filtering, and washing theprecipitate with dry acetonitrile and dichloromethane to remove anyunreacted starting materials, purifying the solid so obtained to giveobtain compound of formula 1 (1a, 1b, 1c and 1d).

[0032] In one embodiment of the invention, the compounds of formula 1are preferably recrystallized from a mixture (1:4) of methanol andacetonitrile.

[0033] Yet another embodiment of the present invention relates tobifunctional molecules of the general formula 1 (1a, 1b, 1c and 1d) andpharmaceutically acceptable derivatives thereof for the photocatalyticcleavage of DNA at G sites of duplex and AG two base bulges containingDNA purely through cosensitization mechanism.

[0034] In another embodiment of the invention, bifunctional molecules ofthe general formula 1 (1a, 1b, 1c and 1d) and pharmaceuticallyacceptable derivatives thereof are used as DNA targeted diagnostic orphototherapeutic agents.

[0035] In another embodiment of the present invention the bifunctionalmolecules of the invention are used for the stabilization of DNAincluding duplex, triplex and quadruplex structures throughintercalation, bisintercalation and groove binding.

[0036] Yet another embodiment of the present invention is the use of thebifunctional molecules of general formula 1 (1a, 1b, 1e and 1d) for thephotocatalytic cleavage of DNA with selectivity at G sites of duplex andAG two base bulges and as probes for these structures.

[0037] Still another embodiment of the present invention is that thebifunctional molecules and derivatives thereof of the general formula 1(1a, 1b, and 1c) are used as photocatalysts for the oxidation of waterin industrial applications.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0038]FIG. 1 shows the fluorescence enhancement of the compound offormula la (wherein n 1, R=—MV²—(CH₂)₃—CH₃ and X=Br) in presence ofvarious concentrations of poly(dA).poly(dT).

[0039]FIG. 2 shows the effect on thermal denaturation temperature of DNAduplex by various concentrations of compound of formula 1a wherein n=1,R=—MV²⁻—(CH₂)₃—CH₃ and X=Br and formula 1b wherein n=1, R=—MV²⁺—CH₂—Acrand X=Br.

[0040]FIG. 3 shows the hypochromocity observed in DNA duplex absorptionin presence of various concentrations of compound of formula la whereinn=1, R=MV²⁻—(CH₂)₃—CH₃ and X=Br and formula 1b wherein n=1,R=—MV²⁺—CH₂—Acr and X=Br.

[0041]FIG. 4 shows the DNA damage profiles showing single strand breaksand various endonuclease-sensitive modifications induced in PM2 DNA bycompound of formula 1a (250 nM, 18 kJ/m²) wherein n=1,R=—MV²⁻—(CH₂)₃—CH₃ and X=Br and at (1 μM, 9 kJ/m²) wherein n=11,R=—MV²⁺—(CH₂)₃—CH₃ and X=Br.

[0042]FIG. 5 shows the time dependence of DNA modifications, singlestrand breaks (SSB) (O) and formamidopyrimidine-DNA glycosylase (FPGprotein) sensitive modifications (Δ) induced in PM2 DNA by compound offormula la wherein n=1, R=—MV²⁺—(CH₂)₃—CH₃ and X=Br (0.25 μM 0° C.) uponUV irradiation with 360 nm light (4.6 kJ/m²).

[0043]FIG. 6 shows the time dependence of DNA modifications, singlestrand breaks (SSB) (O) and formamidoglycosylase (FPG protein) sensitivemodifications (A) induced in PM2 DNA by compound of formula 1a whereinn=11, R=—MV²⁺—(CH₂)₃—CH₃ and X=Br (0.30 μM, 0° C.) upon UV irradiationwith 360 nm light (4.6 kJ/m²).

[0044]FIG. 7 shows the autoradigram of a 10% denaturing polyacrylamidegel showing photocleavage of duplex DNA (10 μM) induced by compound offormula 1a wherein n=1, R=—MV²⁺—(CH₂)₃—CH₃and X=Br and formula 1bwherein n=1, R=—MV²⁺—CH₂—Acr and X=Br with piperidine treatment (90° C.,30 min). Lane 1: DNA (3) (control). Lane 2: DNA (3) and 50 μM ofcompound of formula 1a wherein n=1, R=—MV²⁺—(CH₂)₃—CH₃ and X=Bra Lane 3:DNA (3) and 50 μM of compound of formula 1b wherein n=1, R=—MV²⁺—CH₂—Acrand X=Br. Lane 4: DNA (5) and 50 μM of compound of formula 1a whereinn=1, R=MV²⁺— (CH₂)₃—CH₃ and X=Br. Lane 5: DNA (5) and 50 μM of compoundof formula 1b wherein n=1, R=—MV²⁺—CH₂—Acr and X=Br. Lane 6: DNA (6) and50 μM of compound of formula 1a wherein n=1, R=—MV²⁺—(CH₂)₃—CH₃ andX=Br. Lane 7: DNA (6) and 50 μM of compound of formula 1b wherein n=1,R=MV²⁺—CH₂—Acr and X=Br. The Maxam-Gilbert sequencing lanes are markedwith A/G and T.

[0045]FIG. 8 shows the formation of reduced methyl viologen uponirradiation of compound of formula 1a wherein n=11, R=—MV²⁺—(CH₂)₃—CH₃and X=Br in presence of DNA (sacrificial electron donor), demonstratingthe catalytic properties of viologen linked acridines.

DETAILED DESCRIPTION OF THE INVENTION

[0046] In the present invention, the novel bifunctional moleculesincorporate a DNA intercalator and cosensitizer linked through rigid toflexible spacer groups. The intercalator such as acridine moiety iscapable of absorption in the visible region and acting as anelectron-donor (sensitizer) in the excited state. The cosensitizer,methyl viologen moiety, on the other hand, is a very good electronacceptor and is capable of undergoing groove-binding interactions withDNA. The rigid to flexible spacer groups play a major role incontrolling the rate of electron transfer in these systems through thealteration of distance and relative orientation between the intercalatorand cosensitizer.

[0047] In the present invention, a series of novel viologen linkedacridine, bisacridine and bisacridinium salts of the general formula 1(1a, 1b, 1c and 1d) have been synthesised, having flexible to rigidspacer groups and their photophysical properties examined underdifferent conditions. Further the DNA binding and DNA stabilizationproperties of these molecules have been examined employing calf thymusDNA and synthetic oligonucleotides. The photoactivated DNA cleavingproperties were investigated and the sequence specific cleavage inducedby these compounds by employing plasmid DNA, the synthetic duplex andbase bulge containing DNA sequences were also analysed. Further themechanism of their biological and catalytic activities have beenevaluated.

[0048] As a result, it is observed that compounds of the general formula1 (1a, 1b, 1c and 1d) and their derivatives thereof posses goodsolubility at physiological pH conditions and exhibit good absorptionproperties. Further protonation of the acridine ring leads to theformation of corresponding acridinium salts which show quite differentand interesting photophysical and redox properties. The absorption andfluorescence studies confirm the existence of through bond and throughspace interactions between viologen and acridine moieties. Further,efficient quenching of fluorescence yields was observed and whichindicate the mechanism of quenching is through electron transfermechanism with rates in the order of 10¹⁰.

[0049] Interestingly, these bifunctional molecules exhibit strongbinding with DNA through intercalation and or bisintercalation andgroove binding interactions and with unusually high affinity for thepoly(dA).poly(dT) sequence. Upon excitation, these molecules cleaved DNAvery effectively and with high selectivity at guanine (G) sites andpredominance of 5′-G of GG step. Moreover, these molecules are found tobe very attractive for sequences containing base bulges and cleave uponphotoactivation specifically at the G sites of the AG base bulge. TheDNA cleavage induced by these compounds is purely through the electrontransfer mechanism, where the excited acridine moiety transfers anelectron to methyl viologen (MV²⁺) and lead to the radical cation ofacridine and radical cation of methyl viologen (MV⁺). The radical cationof acridine once formed, can oxidize DNA base at the site of binding andultimately resulted in efficient cleavage of DNA at the G sites, asexpected. Moreover, these molecules are found to be recyclable and actas catalysts in presence of sacrificial electron donors. Therefore, thepresent systems are not only bifunctional with interesting photophysicalproperties but also recyclable and act as effective photoactivated DNAcleaving-and phototherapeutical agents and photocatalysts for theoxidation of water in industrial applications.

[0050] Table 1 shows DNA association constants of bifunctional moleculesbased on viologen linked acridines compound of formula 1a wherein n=1,R=—MV²⁺—(CH₂)₃—CH₃ and X=Br and wherein n=11, R=—MV² —(CH₂)₃—CH₃ andX=Br and bisacridine system (compound of formula 1b wherein n=1,R=—MV²⁺—CH₂—Acr and X=Br) in buffer containing 1 mM EDTA and 2 or 100 mMNaCl.

[0051] Table 2 shows the structures of oligonucleotides used for DNAmelting studies.

[0052] Table 3 shows the structures of oligonucleotides used for DNAcleaving studies.

[0053] The present invention accordingly provides a process for thepreparation of bifunctional molecules represented by compound of formula1 (1a, 1b, 1c and 1d) and derivatives thereof

[0054] Bifunctional molecules of general formula 1 (1a, 1b, 1c and 1d)and pharmaceutically acceptable derivatives thereof are useful for thestabilization of DNA structures including duplex, triplex and quadruplexDNA. The bifunctional molecules of general formula 1 (1a, 1b, 1c and 1d)and pharmaceutically acceptable derivatives thereof are also useful forthe photocatalytic cleavage of DNA at G sites of duplex and AG two basebulges containing DNA purely through cosensitization mechanism. Thenovel molecules of the invention and pharmaceutically acceptablederivatives thereof are useful as DNA targeted diagnostic orphototherapeutic agents. These sensitizers can be used for diagnosis ortreatment of human beings or animals.

[0055] The bifunctional molecules of the invention are used for thestabilization of DNA including duplex, triplex and quadruplex structuresthrough intercalation, bisintercalation and groove binding. Thebifunctional molecules of general formula 1 (1a, 1b, 1c and 1d) can alsobe used for the photocatalytic cleavage of DNA with selectivity at Gsites of duplex and AG two base bulges and hence can be used as probesfor these structures. Bifunctional molecules and derivatives thereof ofthe general formula 1 (1a, 1b, and 1c) are used as photocatalysts forthe oxidation of water in industrial applications.

[0056] The following examples are given by way of illustration andtherefore should not be construed to limit the scope of presentinvestigation.

[0057] Examples 1-4 represent typical synthesis of compounds of generalformula 1 (1a, 1b, 1c and 1d) and Examples 5-11 represent photophysicaland in vitro DNA binding and cleaving properties of bifunctionalviologen linked acridine based molecules.

EXAMPLE 1

[0058] General procedure for the preparation of formulae represented bycompound of formula 1a (wherein Y=—(CH₂)_(n), wherein n=1-11;R=—MV²⁺—(CH₂)_(m)—CH₃2X^(Ξ) or —Pyr²⁺—(CH₂)_(m)—CH₃2X^(Ξ)). A solutionof ω-(acridin-9-yl)-α-bromoalkanes (1-10 mmol) and1-alkyl-4,4′-bipyridinium bromides (1-10 mmol, which in turn wereobtained in 93-98% yields by the reaction of 4,4′-bipyridine with thecorresponding α-bromoalkanes in the molar ratio of 3:1.) in the ratio of1:1 in dry acetonitrile (30-50 mL) was stirred at 20° C. for 10 h. Theprecipitated solid thus obtained was filtered, washed with dryacetonitrile and . dichloromethane to remove any unreacted startingmaterials. The solid was further purified by soxhlet extraction withdichloromethane to give the compound of formula 1a in 70-95% yields.These compounds were recrystallized from a mixture (1:4) of methanol andacetonitrile.

[0059] The physiochemical properties of1-[(acridin-9-yl)methyl]-1′-butyl-4,4′-bipyridinium dibromide (compoundof formula 1a wherein n=1, R=—MV²⁺—(CH₂)₃—CH₃ and X=Br): melting point:260-261° C.; Molecular Weight: MS (FAB), m/z 484 (M⁺Br⁻); ¹H NMR (300MHz, DMSO-d₆): δ=0.91 (t, 3H), 1.26-1.33 (m, 2H), 1.89-1.94 (m, 2H),4.66 (t, 2H), 7.11 (s, 2H), 7.77 (t, 2H), 7.97 (t, 21), 8.31 (d, 2H),8.53 (d, 2H), 8.59 (d, 21), 8.66 (d, 2H), 9.19 (d, 2H), 9.31 (d, 2H);¹³C NMR (75 MHz, DMSO-d₆) δ=149.78-121.41, 61.05, 55.44, 33.01, 19.10,13.66; Nature: Pale yellow powder.

[0060] The physiochemical properties of1-[3-(acridin-9-yl)propyl]-1′-butyl-4,4′-bipyridinium dibromide(compound of formula 1a wherein n=3, R=—MV²⁺—(CH₂)₃—CH₃ and X=Br):melting point: 253-254° C.; Molecular Weight: MS (FAB): m/z 433 (M⁻); ¹HNMR (300 MHz, DMSO-d₆) δ=0.95 (t, 3H), 1.28-1.40 (m, 2H), 1.92-2.02 (m,2H), 2.46-2.51 (m, 4H), 3.95 (t, 2H), 4.74 (t, 2H), 7.79 (t, 2H), 8.05(t, 2H), 8.28 (d, 2H), 8.83-8.86 (m, 6H), 9.45 (d, 2H), 9.57 (d, 2H);¹³C NMR (75 MHz, DMSO-d₆) δ=149.45-124.96, 61.44, 60.73, 33.21, 33.15,24.79, 19.27, 13.80; Nature: Pale yellow powder.

[0061] The physiochemical properties of 1 -[11-(acridin-9-yl)undecyl]-1′-butyl-4,4′-bipyridinium dibromide (compoundof formula 1a wherein n=11, R=—MV²⁺—(CH₂)₃—CH₃ and X=Br): melting point:248-249° C.; Molecular Weight: MS (FAB): m/z 545 (M⁻); ¹H NMR (300 MHz,DMSO-d₆): δ=0.95 (t, 3H), 1.23-1.71 (m, 18H), 1.95-1.98 (m, 4H), 3.65(t, 2H), 4.73 (t, 4H), 7.65 (t, 2H), 7.85 (t, 2H), 8.14 (d, 2H), 8.38(d, 2H), 8.82 (d, 4H), 9.43 (d, 4H); ¹³C NM (75 Mz, DMSO-d₆):148.97-124.71, 61.25, 61.04, 33.07, 31.64, 31.15, 29.68, 29.26, 29:13,28.76, 27.13, 25.7-9, 19.15, 13.71; Nature: Pale yellow powder.

EXAMPLE 2

[0062] General procedure for the preparation of formulae represented bycompound of formula 1b (wherein Y=—(CH₂)_(n), wherein n=1-11;R=—MV²⁺—(CH₂)_(m)—Acr2X^(Ξ) or —Pyr²—(CH₂)_(m)—Acr2X^(Ξ)). A solution ofω-(acridin-9-yl)-α-bromoalkanes (2-10 mmol) and 4,4′-bipyridine (1-5mmol) in the ratio of 2:1 in dry acetonitrile (50-150 mL) was stirred at35° C. for 20h. Precipitated solid was filtered and washed withdichloromethane and acetonitrile. Soxhlet extraction of the solid withdichloromethane gave compound of formula 1b in 65-90% yields.

[0063] The physiochemical properties ofbis-1,1′-[(acridin-9-yl)methyl]-4,4′-bipyridinium dibromide (1b whereinn=1, R=—MV²⁻—CH₂—Acr and X=Br): melting point: >400 ° C.; MolecularWeight MS (FAB): m/z 619 (M⁺Br⁻); ¹H NMR (300 MHz, DMSO-d₆): δ=7.09 (s,4H), 7.77-7.95 (m, 8H), 8.21-8.55 (m, 161), 9.05-9.16 (m, 8); ¹³C NMR(75 MHz, DMSO- d₆) δ=150.85-124.51, 58.52; Nature: Pale yellow powder.

[0064] The physiochemical properties ofbis-1,1′-[3-(acridin-9-yl)propyl]-4,4′-bipyridinium dibromide (1bwherein n=3, R=—MV²⁺—(CH₂)₃—Acr and X=Br): melting point: 223-224° C.;Molecular Weight: MS (FAB): m/z 596 (M⁺); ¹H NMR (300 MHz, D₂O):δ=2.50-2.60 (m, 4H), 3.95 (t, 4H), 4.9 (t, 4H), 7.79-7.83 (m, 6H),8.01-8.07 (m, 8H), 8.26 (d, 2H), 8.46-8.48 (m, 4H), 8.69 (d, 2H), 8.93(d, 2H); ¹³C NMR (75 MHz, DMSO-d₆): δ=150.82-121.86, 60.26, 32.36,24.30; Nature: Pale yellow powder.

[0065] The physiochemical properties ofbis-1,1′-[11-(acridin-9-yl)undecyl]4,4′-bipyridinium dibromide (1bwherein n=11, R=—MV²⁺—(CH₂)₁₁—Acr and X=Br): melting point: 152-153° C.;Molecular Weight: MS (FAB): m/z 820 (M+); ¹H NMR (300 MHz, DMSO-d₆):δ=1.22-1.94 (m, 36H), 3.63 (t, 4H), 4.64 (t, 4H), 7.65 (t, 4H), 7.85(t,4H), 8.04 (d, 4H), 8.15 (d, 4H), 8.37 (d, 2H), 8.63 (d, 2H), 8.87(d,2H), 9.24 (d, 2H): 1³C NMR (75 MHz, DMSO-d₆): δ=151.36-122.30, 60.83,31.63, 31.07, 29.69, 29.26, 29.11, 28.75, 27.13, 25.79; Nature: Paleyellow powder.

EXAMPLE 3

[0066] Synthesis of compound of formula 1c (wherein Y=ortho or paratolyl; R=MV²⁻—(CH₂)_(m)—CH₃2X^(Ξ) or —Pyr²⁺—(CH₂)_(m)—CH₃2X^(Ξ)). Asolution of 9-(2-bromomethylphenyl)acridine (1-5 mmol) and1-butyl-4,4′-bipyridinium bromide (1-5 mmol) in the ratio of 1:1 in dryacetonitrile (30-120 mL) was stirred at 50° C. for 15h. The precipitatedsolid thus obtained was filtered, washed with dry acetonitrile anddichloromethane to remove any unreacted starting materials. The solidwas further purified by soxhlet extraction with dichloromethane to givecompound of formula 1c (wherein n=1, R=—MV²⁺—(CH₂)₃—CH₃, X=Br andacridine is at the ortho position) in 35-55% yield and the product wasfurther purified by recrystallization from ethyl acetate. Similarly, thereaction of 9-(4-bromomethyl phenyl)acridine (1-5 mmol) and1-butyl-4,4′-bipyridinium bromide (1-5 mmol) in the ratio of 1:1 in dryacetonitrile (30-120 mL) gave compound of formula 1c (wherein n=1,R=—MV²⁻—(CH)₃—CH₃, X=Br and acridine is at the para position) in 55-70%yield and the product was further purified by recrystallization fromethyl acetate.

[0067] The physiochemical properties of1-[(2-(acridin-9-yl)-1-methyl)phenyl]-1′-butyl-4,4′-bipyridiniumdibromide (1c wherein n=1, R=—MV²⁺—(CH₂)₃—CH₃, X=Br and acridine is atthe ortho position): melting point: 224-225° C.; Molecular Weight: MS(FAB): m/z: 561 (M⁺Br⁻); ¹H NMR (300 MHz, DMSO-d₆): δ=0.95 (t, 3H),1.31-1.39 (m, 2H), 1.95-2.00 (m, 2H), 4.73 (t, 2H), 5.56 (s, 2H), 7.21(d, 2H), 7.42 (t, 21), 7.5 (d, 2H), 7.75-7.89 (m, 4H), 8.20-8.23 (m,4H), 8.41 (d, 2H), 8.55 (d, 2H), 9.4 (d, 2H); ¹³C NMR (75 MHz, DMSO-d₆):δ=148.49-124.24, 62.01, 60.64, 32.67, 18.78, 13.35; Nature: Yellowpowder.

[0068] The physiochemical properties of1-[(4-(acridin-9-yl)-1-methyl)phenyl]-1′-butyl4,4′-bipyridiniumdibromide (1c wherein n=1, R=—MV²⁺—(CH₂)₃—CH₃, X=Br and acridine is atthe para position): melting point: 268-269° C.; Molecular Weight: MS(FAB): m/z 561 (M⁺Br⁻); ¹H NMR (300 MHz, DMSO-d₆): δ=0.94 (t, 3H),1.31-1.38 (m, 2H), 1.90-1.95 (m, 2H), 4.72 (t, 2H), 6.18 (s, 2H), 7.57-7.65 (m, 6H), 7.88-7.92 (m, 4H), 8.24 (d, 2H), 8.86 (d, 2H), 8.88 (d,2H), 9.43 (d, 2H), 9.69 (d, 2H); ¹³C NMR (75 MHz, DMSO-d₆):δ=149.23-124.22, 62.86, 60.63, 32.70, 18.76, 13.32; Nature: Yellowpowder.

EXAMPLE 4

[0069] General procedure for the preparation of formulae represented bycompound of formula 1d (wherein Y=—(CH₂)_(m), wherein n=1-10;R=—Acr⁺—R¹X^(Ξ)/2X^(Ξ) wherein N in the acridine ring main ring is alsoquarternised by alkyl group. R¹=—(CH₂)_(m)—CH₃ and—(CH₂)_(m)—C₆H₄—(CH₂)_(m)—(para) wherein m=0-13: A solution ofα,ω-bis(9-acridinyl)alkane (1-5 mmol) and alkyl halide (3-15 mmol) inthe ratio of 1:3 in dry acetonitrile (30-150 mL) was refluxed for 8-24h. The precipitated solid was filtered and washed with dry acetonitrileand dichloromethane in small portions to remove unreacted startingmaterials. The solid was further purified by recrystallization from amixture (1:4) of ethyl acetate and acetonitrile to give compound offormula Id in 65-95% yields.

[0070] The physiochemical properties of compound of formula 1d (whereinn=5, R=—Acr⁻—CH₃ and X=I): melting point: 222-223 ° C.; MolecularWeight: MS (FAB): m/z 561 (MBr) ¹H NMR (300 MHz, DMSO-d₆): δ=1.88 (m,6H), 3.99 (t, 4H), 4.82 (s, 6H), 8.01 (t. 4H), 8.35 (t, 4H), 8.78 (d,4H), 8.87 (d, 4H); ^(—)C NMR (75 MHz, DMSO-d₆): δ=164.38-20.01, 32.60,32.22, 30.19, 29.37; Nature: Yellow powder.

[0071] The physiochemical properties of compound of formula 1d (whereinn=10, R=—Acr⁺—CH₃ and X=I): melting point: 230-232° C.; ¹H NMR (300 MHz,DMSO-d₆): δ=1.26-1.74 (m, 16H), 3.97 (t, 4H), 4.82 (s, 6H), 8.03 (t,4H), 8.43 (t, 4H), 8.77 (d, 4H), 8.9 (d, 4H); ¹³C NMR (75 MHz, DMSO-d₆):δ=164.65-120.03, 33.75, 33.05, 29.94, 29.44, 29.32, 24.97; Nature:Yellow powder.

EXAMPLE 5

[0072] DNA binding efficiency. DNA binding affinities ofacridine-viologen bifunctional molecules represented by compound offormula 1a and 1d were analyzed using calf thymus DNA, in NaCl buffer atdifferent salt concentrations (2 mM and 100 mM). Test solutionscontaining different concentrations of calf thymus DNA in NaCl bufferwere incubated at room temperature for one hour to complete thecomplexation and were analyzed using absorption and fluorescencetechniques. Strong hypochromicity in absorption and effective quenchingof fluorescence emission yields of viologen linked acridines wereobserved in presence of DNA. The binding constants of viologen linkedacridines with DNA were determined according to the reported procedures.References may be made to Peacocke, A. R.; Skerrett, J. N. H. Trans.Faraday Soc. 1956, 52, 261; McGhee, J. D.; von Hippel, P. H. J. Mol.Biol. 1974, 86, 469; Scatchard, G. Ann. N.Y. Acad. Sci 1949, 51, 660;Adam, W.; Cadet, J.; Dall'Acqua, F.; Epe, B.; Ramaiah, D.; Saha-Moller,C. R. Angew. Chem. Int. Ed. Engl. 1995, 34, 107). These moleculesexhibited appreciable binding affinity (in the order of 10⁶) for calfthymus DNA and were found to be one order less at higher saltconcentration (Table 1). Further fluorescence lifetimes of thesecompounds were also examined in presence and absence of DNA. TABLE 1Compound Ionic strength K (M⁻¹) 1a wherein n = 1; R = -MV²⁺—(CH₂)₃—  2mM NaCl 9.24 × 10⁵ CH₃; X = Br 100 mM NaCl 1.01 × 10⁵ 1a wherein n = 11;R = -MV²⁺—(CH₂)₃—  2 mM NaCl 5.24 × 10⁶ CH₃; X = Br 100 mM NaCl 1.64 ×10⁵ 1b wherein n = 1; R = -MV²⁺—CH₂-Acr;  2 mM NaCl 1.30 × 10⁶ X = Br100 mM NaCl 3.90 × 10⁵

[0073] These results indicate that the planar acridine ring canintercalate between the base pairs of calf thymus DNA in a positionperpendicular to the helix axis. At the same time, the viologen moietyinteracts electrostatically with the phosphate backbone of the DNA. Thestrong dependence of the binding constants on ionic strength of thebuffer medium is a strong indication that these systems interact withDNA through intercalation as well as by groove binding, indicating theirbifunctional in character.

EXAMPLE6

[0074] Demonstration of special affinity for poly(dA).poly(dT) sequence.In order to have a better understanding on the binding site of acridinechromophore in viologen linked acridines in DNA, the absorption andfluorescence properties of these molecules were examined in presence ofvarious polynucleotides. The gradual addition of poly(dA).poly(dT) tothe buffered solutions of viologen linked acridines led to a gradualdecrease in absorption intensity with a strong enhancement in theirfluorescence emission yields (FIG. 1). However, no significant changeswere observed when a solution of poly(dG).poly(dC) was added. Theseresults indicate the fact that the viologen linked acridines examinedherein posses special affinity for A.T sequence and hence can havepotential applications as probes for the detection of such sequences inDNA.

EXAMPLE 7

[0075] Enhancement in thermal stability of DNA. Intercalation of smallligands into DNA duplex is known to increase the DNA melting temperature(T_(m)), i.e. the temperature at which the double helix denatures intosingle stranded DNA (Gasparro, F. P. Psoralen DNA photobiology, CRCPress Inc.; Boca Raton, Fla., 1988; Patel, D. J.; Cannel, J. Proc. Natl.Acad. Sci. USA 1976, 73, 674). To examine the effect of the presentsystems on the thermal denaturation of DNA, experiments were carried outemploying calf thymus DNA and synthetic oligonucleotides listed in Table2. In 10 mM phosphate TABLE 2 DNA sequence DNA(1) 5′-CGT GGA CAT TGC ACGGTA C-3′ DNA(2) 5′-GTA CCG TGC AAT GTC CAC G-3′

[0076] buffer, both compound of formula 1a and 1b (wherein Y—(CH₂)_(n),wherein n=1-11; R=—MV²⁺—(CH₂)_(m)—CH₃2X^(Ξ) or—Pyr²⁺—(CH₂)_(m)—CH₃2X^(Ξ) and wherein Y=—(CH₂)_(n), wherein n=1-11;R=—MV²⁺—(CH₂)_(m)—Acr2X^(Ξ) or —Pyr²⁺—(CH₂)_(m)—Acr2X^(Ξ) respectively)were found to stabilize the DNA and the extent of stabilizationincreases with the increasing in concentration of viologen linkedacridines as shown in FIG. 2. At all concentrations of the ligand onlyone transition temperature was observed, in each case, therebyindicating that only one type of binding with DNA is responsible forsuch behavior. The extent of stabilization was found to be nearly 20° C.in the case of at 40 μM of compound of formula 1a (wherein Y=—(CH₂)_(n),wherein n=1-11; R=—MV²⁺—(CH₂)_(m)—CH₃2X^(Ξ) or—Pyr²⁺—(CH₂)_(m)—CH₃2X^(Ξ)), whereas 20 μM of compound of formula 1b(wherein Y=—(CH₂)_(n), wherein n=1-11; R=—MV²⁺—(CH₂)_(m)—Acr2X^(Ξ) or—Pyr²⁺—(CH₂)_(m)—Acr2X^(Ξ))showed nearly 18° C. stability.

[0077] In addition to the significant thermal stability, unusually largehypochromicity (around 80%) was observed upon binding of these moleculeswith DNA as shown in FIG. 3. These results indicate the existence ofstrong π-stacking interactions between the ligand molecules and DNAbases. This result in, only partial separation of DNA strands onmelting, leading to a decrease in absorbance change. The largestabilization and significant hypochromicities offered by the structuresof formula 1a and 1b prove further their strong interaction with DNA andpotential use in biology for the detection of various DNA structures,stabilization of triplex and G-quadruplex structures.

EXAMPLE 8

[0078] Demonstration as photoactivated DNA cleaving agents. Cleavage ofplasmid DNA was followed by monitoring the conversion of supercoiled(Form I) to open circular relaxed (Form II) and linear (Form III).Plasmid DNA cleavage is a very sensitive technique and when combinedwith several repair endonucleases, it can serve as a kind of fingerprintof the species directly responsible for the damage. Induction of onesingle strand break (SSB) by a compound converts Form I to Form II andthe quantification of which indicates its efficiency of DNA cleavage. ADNA relaxation assay was used to quantify SSB and endonuclease-sensitivemodifications (Epe, B.; Helger, J.; Wild, D. Carcinogenesis 1989, 10,2019 and Epe, B.; Mftzel, P.; Adam, W. Chem. Biol. Interactions 1988,67, 149). This assay makes use of the fact that Form I when converted byeither a single strand break (SSB) or the incision by a repairendonuclease leads to Form II, which migrates separately in agarose gelelectrophoresis.

[0079] Phosphate-buffered (pH 7.0), air-saturated solutions of PM2 DNA(10 μg/mL) at 0° C. were irradiated with 360 nm near-UV irradiation inthe presence of various concentrations of acridine-viologen bifunctionalmolecules represented by compound of formula 1a and 1b (whereinY=—(CH₂)_(n), wherein n=1-11; R=—MV²⁺—(CH₂)_(m)—CH₃2X^(Ξ) or—Pyr²⁺—(CH₂)_(m)—CH₃2X^(Ξ) and wherein Y=—(CH₂)_(n), wherein n=1-11;R=—MV²⁺—(CH₂)_(m)—Acr2X^(Ξ) or —Pyr²⁺—(CH₂)_(m)—Acr2X^(Ξ) respectively).Subsequently, the DNA was analyzed for the following types ofmodifications: (i) DNA single and double strand breaks; (ii) sites ofbase loss (AP sites) recognized by exonuclease III; (iii) basemodifications plus AP sites sensitive to the T4 endonuclease V; (iv)base modifications plus AP sites sensitive to the endonuclease III and(v) base modifications plus AP sites sensitive toformamidopyrimidine-DNA glycosylase (FPG protein). DNA damage profileinduced by the acridine-viologen bifunctional molecules are presented inFIG. 4.

[0080] It is evident from the damage profiles that both the compoundsinduced very few AP sites and few modifications sensitive toendonuclease III, but a large number of base modifications sensitive toFPG protein were observed. Further, no significant DNA damage wasobserved either by irradiation of PM2 DNA alone or in the dark inpresence of viologen linked acridines at the highest concentrations,thereby indicating that the damage observed is purely initiated by thephotoactivation of these compounds. Hence these compounds can be used asefficient photoactivated DNA cleaving agents.

EXAMPLE 9

[0081] Efficiency of DNA cleavage. Since the major damage induced by theacridine-viologens is recognized by the FPG protein (FIG. 4), we haveinvestigated the effect of irradiation time and concentration of thesesystems on the formation of FPG sensitive modifications and SSB. FIG. 5and FIG. 6 show the irradiation time dependent formation ofsingle-strand breaks (SSB) and FPG sensitive modifications induced bythe acridine-viologen bifunctional derivatives compound of formula 1a(wherein n=1, R=—MV²⁺—(CH₂)₃—CH₃ and X=Br and wherein n=11,R=—MV²⁺—(CH₂)₃—CH₃ and X=Br), respectively. As can be seen from thesefigures the damage sensitive to FPG protein increases, in each case,with increase in the time of irradiation, indicating the catalyticproperty of these compounds. No significant increase in the generationof SSB was observed, even after the irradiation for 30 min. Similarly,increase in DNA damage was observed with the increase in concentrationas shown in the inset of FIG. 6.

[0082] These results clearly demonstrate that the acridine-viologenbifunctional derivatives induce large number of base modificationssensitive to the repair endonuclease FPG protein, with little damagerecognized by repair endonuclease III. FPG protein is known to recognizemodifications such as 8-oxoguanosine and formamido-pyrimidines,ring-opened products of purines (Boiteux, S.; Gajewski, E.; Laval, J.;Dizdarough, M. Biochemistry, 1992, 31, 106). , Both 8-oxoguanosine andformamidopyrimidines can be generated in DNA by hydroxyl radicals (Epe,B.; Haring, M.; Ramaiah, D.; Stopper, H.; Abou-Elzahab, M.; Adam, W.;Saha-Moller, C. R. Carcinogenesis 1993, 14, 2271; Epe, B.; Pflaum, M.;Haring, M.; Hegler, J.; Rudiger, H. Toxicol Lett. 1993, 67, 57), singletoxygen and by electron transfer mechanism (von Sonntag, C. The ChemicalBasis of Radiation Biology; Taylor and Francis, London, 1987).

[0083] The involvement of both hydroxyl radicals and singlet oxygen canbe ruled out since the damage profiles shown in FIG. 4 are differentfrom those induced by the ionizing radiation and disodium salt of1,4-etheno-2,3-benzoxlioxin-1,4-dipropanoic acid (Aruoma, O. I.;Halliwell, B.; Dizdaroglu, M. J. Biol. Chem. 1989, 264, 13024; Muller,E.; Boiteux, S.; Cunningham, R. P.; Epe, B. Nucl. Acids Res.1990, 18,5969). In addition, DNA cleavage studies were examined in presence ofvarious additives. Results of these studies indicate thatacridine-viologen bifunctional molecules can be used as reagents forinduction of DNA damage purely through photoinduced electron transferparticularly for the modification (oxidation) of guanine base in DNA.

EXAMPLE 10

[0084] Demonstration of cleavage at guanine (G), preferential cleavageof 5′-G of GG sequence and G of a AG bulge. In order to examine theselectivity in cleavage and also to find out whether if there is anypreferential reaction of the viologen linked acridines towards basebulges, we have analyzed the cleavage reactions using a few end labeledsynthetic oligonucleotides (Table 3) by polyacrylamide gelelectrophoresis (PAGE). TABLE 3 DNA sequence DNA(3) 5′-*CAC TGG CTT TTCGGT GCA T-3′ DNA(4) 5′-ATG CAC CGA AAA GCC AGT G-3′ DNA(5) 5′-*CAC TGGCTT CCT TCG GTG CAT-3′ (CC-bulge) DNA(6) 5′-*CAC TGG CTT AGT TCG GTGCAT-3′ (AG-bulge)

[0085] Oligonucleotides DNA(3), DNA(5) (CC-bulge) and DNA(6) (AG-bulge)were radiolabeled at 5′-OH using [γ-³²P]ATP and bacterial T4polynucleotide kinase according to the standard procedure (Sambrook, J.;Fritsch, E. F.; Maniatis, T. Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York, 1989) and hybridized withDNA(4), incubated with various concentrations of viologen linkedacridines and irradiated with a light source in microcentrifuge tubes ina Rayonet Photoreactor (RPR) containing eight 350 nm lamps according thereported procedure (Ramaiah, D.: Kan, Y.; Koch, T.; Qrum, H.; Schuster,G. B. Proc. Natl. Acad Sci. USA 1998, 95, 12902; Ramaiah, D.; Koch, T.;Qrum, H.; Schuster, G. B. Nucl. Acids Res. 1998, 26, 3940). Afterirradiation, the samples were precipitated with buffer, washed two timeswith cold 70% aqueous ethanol, treated with hot piperidine for 30 min at90° C., lyophilized under vacuum and analyzed by PAGE. Maxam-GilbertA/G, T and C- specific reactions were performed by routine protocols(Maxam, A. M.; Gilbert, W. Methods in Enzymol. 1980, 65, 499; Ramaiah,D.: Kan, Y.; Koch, T.; Qrum, H.; Schuster, G. B. Proc. Natl. Acad. Sci.USA 1998, 95, 12902).

[0086] The selectivity of cleavage pattern of 5′-labeled DNA (3), DNA(5) and DNA (6), caused by the bifunctional acridine-viologenderivatives of compound of formula 1a and 1b (wherein Y=—(CH₂)_(n),wherein n=1-11; R=MV²⁺—(CH₂)_(m)—CH₃2X^(Ξ) or —Pyr²⁺—(CH₂)_(m)—CH₃2X^(Ξ)and wherein Y=—(CH₂)b, wherein n=1-11; R=—MV²⁺—(CH₂)_(m)—Acr2X^(Ξ) or—Pyr²⁺—(CH₂)_(m)—Acr2X^(Ξ) respectively) is shown in FIG. 7. Both thesecompounds were found to cleave DNA(3), at the GG sites with asignificant preference for the 5′-G over the 3′-G (lanes 2 and 3 of FIG.7). Small amount of cleavage was also observed at the G site Similarobservations were made with DNA(5) (CC-bulge) by both of compound offormulae 1a and 1b (lanes 4 and 5 of FIG. 7). Practically no cleavage atthe CC bulge site was observed. Irradiation of DNA(6) (AG-bulge) inpresence of compound of formula 1a and 1b (wherein Y=—(CH₂)_(n), whereinn=1-11; R=—MV²⁺—(CH₂)_(m)—CH₃2X^(Ξ) or —Pyr²⁻—(CH₂)_(m)—CH₃2X^(Ξ) andwherein Y=—(CH₂)_(n), wherein n=1-11; R=—MV²⁺—(CH₂)_(m)—Acr2X^(Ξ) or—Pyr²⁻—(CH₂)_(m)—Acr2X^(Ξ) respectively), caused remarkably selectivecleavage at the G of the AG bulge (lanes 6 and 7 of FIG. 7). Compound offormula 1a (wherein Y=—(CH₂)_(n), wherein n=1-11;R=—MV²⁺—(CH₂)_(m)—CH₃2X^(Ξ) or —Pyr²⁺—(CH₂)_(m)—CH₃2X^(Ξ)) was found tobe more efficient in cleaving the duplex DNA structures and alsoduplexes containing the base bulges. These results indicate that thesemolecules can be used for the photoactivated selective cleavage of 5′-Gof the GG sites and G of the AG two base bulges in DNA and also fortheir recognition.

EXAMPLE 11

[0087] Demonstration of catalytic activity. Since the systems underinvestigation posses high DNA association constants and found to cleaveefficiently at G sites in DNA duplex and base bulges upon irradiation,we further demonstrated their catalytic properties so that they can havepotential applications in biology and industry. Direct laser excitationof the viologen linked acridine of formula 1a, 1b and 1c (whereinY=—(CH₂)₆, wherein n=1-11; R=—MV²⁺—(CH₂)_(m)—CH₃2X^(Ξ) or—Pyr²⁺—(CH₂)_(m)—CH₃2X^(Ξ) wherein Y=—(CH₂)_(n), wherein n=1-11;R=—MV²⁺—(CH₂)_(m)—Acr2X^(Ξ) or —Pyr²⁺—(CH₂)_(m)—Acr2X^(Ξ); and whereinY=ortho or para tolyl; R=—MV²⁺—(CH₂)_(m)—CH₃2X^(Ξ) or—Pyr²⁺—(CH₂)_(m)—CH₃2X^(Ξ) respectively) in water or buffer showed noabsorptions assignable to transient intermediates. However, when thelaser excitation of viologen linked acridine of formula 1a (whereinY=—(CH₂)_(n), wherein n=1-11; R=—MV²⁺—(CH₂)_(m)—CH₃2X^(Ξ) or—Pyr²⁺—(CH₂)_(m)—CH₃2X^(Ξ) was carried out in water or buffer in thepresence of external electron donors such as N,N-dimethylaniline (10 mM)or guanosine (1.8 mM) a transient species with absorption maxima at 395and 610 nm, was observed. The spectral features of this transient weresimilar to those of methyl viologen radical cation (MV⁻), reported inthe literature (Watanabe, T.; Honda, K. J. Phys. Chem. 1982, 86, 3661;Kelly, L. A.; Rodgers, M. A. J. J. Phys. Chem. 1994, 98, 6377).Similarly, laser excitation of viologen linked acridine of formula 1a(wherein Y=—(CH₂)_(n), wherein n=1-11; R=MV²⁻—(CH₂)_(m)—CH₃2X^(Ξ) or—Pyr²⁺—(CH₂)_(m)—CH₃2X^(Ξ)) in the presence of DNA in water or buffer,gave a transient species assignable to the reduced methyl viologen (FIG.8).

[0088] These results clearly show that the excited acridine chromophoretransfers an electron to the viologen moiety leading to the formation ofradical cation of acridine and reduced methyl viologen The radicalcation of acridine once formed, oxidizes the electron donor present inthe medium (the formation of methyl viologen radical cation (MV⁺) isfacilitated by external sacrificial electron donors) and reverts back toacridine for reabsorption of photons. The reduced methyl viologen can inturn transfer an electron to the molecular oxygen thereby reverting backto methyl viologen and generating superoxide radical anion. Therefore,these bifunctional molecules and derivatives thereof can function ascatalytic photoactivated DNA cleaving agents in presence of sacrificialelectron donors and as catalysts for the oxidation of water underappropriate conditions to generate hydrogen in industrial applications.

[0089] Bifunctional molecules of the present invention posses excellentproperties of a photosensitizer for phototherapeutical as well ascatalytic photoactivated DNA cleaving and industrial applications. Themain advantages of the present systems include:

[0090] 1. Compounds represented by formula 1 (1a, 1b, 1c and 1d) arepure single substances.

[0091] 2. Their synthetic methodology is very economical.

[0092] 3. They are very stable, highly soluble in aqueous medium andexist in the neutral form under physiological conditions.

[0093] 4. These compounds posses good absorption properties and are verystable in the dark and under irradiation conditions.

[0094] 5. They constitute an effective donor-acceptor system and theirredox properties can be tuned as a function of pH and spacer group.

[0095] 6. They form a novel class of compounds with high affinity to DNAand interact with DNA through intercalation, bisintercalation and groovebinding.

[0096] 7. These systems stabilize various structures of DNA includingduplex DNA, base bulges, triplex and quadruplex DNA structures.

[0097] 8. These systems can be easily covalently linked tooligonucleotides for the stabilization of triplex DNA and also for theselective photocleavage of DNA.

[0098] 9. These systems posses special affinity for A.T sequence andhence can have potential applications as probes for the detection ofsuch sequences in DNA

[0099] 10. They form a novel class of compounds, which cleave DNA in acatalytic way under irradiation conditions and act as catalyticphotoactivated DNA cleaving agents.

[0100] 11. They cleave DNA purely through the photoinduced electrontransfer mechanism and selectively at guanine sites and with excellentselectivity at 5′-G of GG sequence in DNA.

[0101] 12. They cleave duplex DNA containing AG base bulges selectivelyat G sites and hence act as probes for the detection of AG bulgecontaining DNA sequence.

[0102] 13. These systems form a novel class of photosensitizers, whichupon irradiation in presence of external donors results in an effectivecharge separation and hence these systems and derivatives thereof canact as photocatalysts in industrial applications including in thephotoinduced hydrogen generation from water.

1 6 1 19 DNA Artificial Sequence synthetic oligonucleotide 1 cgtggacattgcacggtac 19 2 19 DNA Artificial Sequence synthetic oligonucleotide 2gtaccgtgca atgtccacg 19 3 19 DNA Artificial Sequence syntheticoligonucleotide 3 cactggcttt tcggtgcat 19 4 19 DNA Artificial Sequencesynthetic oligonucleotide 4 atgcaccgaa aagccagtg 19 5 21 DNA ArtificialSequence synthetic oligonucleotide 5 cactggcttc cttcggtgca t 21 6 21 DNAArtificial Sequence synthetic oligonucleotide 6 cactggctta gttcggtgca t21

We claim:
 1. Viologen linked acridine based molecule of the generalformula 1 (1a, 1b, 1c and 1d) below

1a wherein Y=—(CH₂)_(m)— wherein n=1-11R=—MV²⁺—(CH₂)_(m)—CH₃2X^(Θ)or—Pyr²⁺—(CH₂)_(m)—CH₃2X^(Θ) wherein m=1-13 1b wherein Y=—(CH₂)_(n)—wherein n=1-11R=—MV²⁺—(CH₂)_(m)—Acr 2X^(Θ) or—Pvr²⁺—(CH₂)_(m)—Acr 2X^(Θ)wherein m=1-11 1c wherein Y=ortho or paratolylR=—MV²+—(CH₂)_(m)—CH₃2X^(Θ) or—Pyr²⁺(CH₂)_(m)—CH₃2X^(Θ) whereinm=1-13 1d wherein Y=—(CH₂)_(n)— wherein n=1-10R=—Acr⁺—R¹X^(Θ)/2X^(Θ)wherein N in the acridine main ring is also quarternised by alkyl groupR¹=—(CH₂)_(m)—CH₃ and —(CH₂)_(m)—C₆H₄—(CH₂)_(m)—(para),wherein m=0-13

and a pharmaceutically acceptable derivative thereof.
 2. A process forthe preparation of viologen linked acridine based molecule of thegeneral formula 1 below

1a wherein Y=—(CH₂)_(m)— wherein n=1-11R=—MV²⁺—(CH₂)_(m)—CH₃2X^(Θ)or—Pyr²⁺—(CH₂)_(m)—CH₃2X^(Θ) wherein m=1-13 1b wherein Y=—(CH₂)_(n)—wherein n=1-11R=—MV²⁺—(CH₂)_(m)—Acr 2X^(Θ) or—Pvr²⁺—(CH₂)_(m)—Acr 2X^(Θ)wherein m=1-11 1c wherein Y=ortho or paratolylR=—MV²+—(CH₂)_(m)—CH₃2X^(Θ) or—Pyr²⁺(CH₂)_(m)—CH₃2X^(Θ) whereinm=1-13 1d wherein Y=—(CH₂)_(n)— wherein n=1-10R=—Acr⁺—R¹X^(Θ)/2X^(Θ)wherein N in the acridine main ring is also quarternised by alkyl groupR¹=—(CH₂)_(m)—CH₃ and —(CH₂)_(m)—C₆H₄—(CH₂)_(m)—(para),wherein m=0-13

comprising forming a solution of ω-(acridin-9-yl)-α-bromoalkanes and/orI-alkyl-4,4′-bipyridinium bromides in dry acetonitrile in the ratio of1:1, stirring the above solution at a temperature in the range of 20-50°C. for a time period in the range between 8-24 h to obtain aprecipitate, filtering, and washing the precipitate with dryacetonitrile and dichloromethane to remove any unreacted startingmaterials, purifying the solid so obtained to give obtain compound offormula 1 (1a, 1b, 1c and 1d).
 3. A process as claimed in claim 2wherein the compound of formula 1 is recrystallized from ethyl acetate,methanol, dry acetonitrile, dichloromethane or any mixture thereof
 4. Aprocess as claimed in claim 3 wherein the compound of formula 1 isrecrystallized from a mixture of methanol and acetonitrile in a ratio of1:4.
 5. Use of the viologen linked acridine based molecule of thegeneral formula 1 as phototherapeutical agents.